1. Field of the Invention
The present invention relates to a method for producing a dry reagent that is used in analysis of components that are contained in a liquid sample, and to a dry reagent and an analysis tool that uses the dry reagent.
2. Description of the Related Art
Nicotinamide coenzymes (for instance, β-NAD+, β-NADH, β-NADP+ and β-NADPH) are widely used as detection reagents among reagents for measuring, in accordance with enzymatic methods, the amount of components, for instance AST (aspartate aminotransferase) or LDH (lactate dehydrogenase) contained in a liquid sample. For instance, Japanese Unexamined Patent Application Publication No. H8-248028 discloses a liquid reagent that contains a nicotinamide coenzyme and that is used for measuring components in a liquid sample. In this liquid reagent, the increase or decrease of a nicotinamide coenzyme is measured in accordance with a transmission method that utilizes light of a wavelength (340 nm) in the ultraviolet region.
There have been proposed analysis tools for measuring, in a simpler manner, the amount of components in liquid sample, wherein the analysis tool is provided with a detection reagent in the form of a dry reagent that contains a nicotinamide coenzyme. For instance, Japanese Examined Patent Application Publication No. H5-60360 discloses an analysis tool wherein multiple gelatin layers are formed on a substrate, and β-NAD+, which is one nicotinamide coenzyme, is contained in the layers. The analysis tool does not detect directly β-NADH, which results from reduction of β-NAD+; instead, the analysis tool comprises a formazan dye precursor and an electron transfer agent, as a β-NADH detection agent. Specifically, diaphorase is used as the electron transfer agent and nitrotetrazolium blue (hereafter NTB) is used as the formazan dye precursor. The change in color of NTB is detected in accordance with a reflection method that utilizes light of a wavelength in the visible region.
WO 2009/090756 discloses an analysis tool that is made up of a sample supply port through which a liquid sample is supplied into the analysis tool, a measurement chamber in which the liquid sample is measured, and a flow channel that communicates the sample supply port with the measurement chamber. The flow channel and the measurement chamber are porous plate members that are formed through stacking of a plurality of plate members at least one of which has air permeability. Transport of the liquid sample in this analysis tool is accomplished not by capillarity, but, for instance, by pressure that is applied from the sample supply port. A dry reagent is disposed in the measurement chamber of the analysis tool. In this dry reagent as well, β-NAD+ is used as a detection reagent. Herein, β-NADH resulting from reduction of β-NAD+ is not detected directly; instead, the dry reagent comprises a β-NADH detection agent that detects β-NADH resulting from reduction of β-NAD+. Diaphorase is used as an electron transfer agent, and WST-4, which is a water-soluble tetrazolium salt, is used as a formazan dye precursor. The color change of WST-4 is detected in accordance with a transmission method that uses a wavelength in the visible region.
Japanese Unexamined Patent Application Publication No. H10-197526 discloses an analysis tool comprising a transparent substrate and a transparent cover, and an analyzer for measuring the analysis tool. In this analysis tool, a measurement chamber having a dry reagent disposed therein, and a capillary flow channel for transport of a liquid sample to the measurement chamber, are formed through bonding of the transparent substrate and the transparent cover to each other. The liquid sample that is transported in the measurement chamber dissolves the dry reagent. In the analyzer, a change in color that arises as a result of a reaction between the detection reagent that is contained in the dry reagent and a specific component in the liquid sample is measured in accordance with a transmission method that utilizes light of the visible region. Measurements in the analysis tool are performed with the liquid sample sealed. Contamination in the analyzer can be prevented accordingly. Therefore, the interior of the analyzer need not be cleaned. The analyzer is made smaller through improvements in an optical system. This dry reagent does not comprise a nicotinamide coenzyme as a detection reagent.
However, the above conventional technologies leave room for improvement as regards the features below.
Enzymatic methods that utilize nicotinamide coenzymes are widely used, as disclosed in Japanese Unexamined Patent Application Publication No. H8-248028, Japanese Examined Patent Application Publication No. H5-60360, and WO 2009/090756. However, lack of interchangeability in the measured values may preclude comparing patient data and may interfere with diagnosis. Therefore, the International Federation of Clinical Chemistry (IFCC) issues standard measurement methods (IFCC-recommended methods), at the international level, while the Japan Society of Clinical Chemistry (JSCC) publishes JSCC-recommended methods in Japan. A major characterizing feature of these recommended methods is that the amount of nicotinamide coenzymes are measured directly at 340 nm, which is a wavelength in the ultraviolet region. Such recommended methods are used in the liquid reagent disclosed in Japanese Unexamined Patent Application Publication No. H8-248028, but are also preferably used in analysis tools for easy measurement, such as those disclosed in Japanese Examined Patent Application Publication No. H5-60360 and WO 2009/090756.
However, measurements that rely on light of a wavelength (340 nm) in the ultraviolet region are fundamentally difficult to perform in reflection methods. Therefore, analysis tools that work on the basis of a reflection method such as the one disclosed in Japanese Examined Patent Application Publication No. H5-60360 rely necessarily on a measurement principle that utilizes light of a visible region, unlike the abovementioned recommended methods.
In the analysis tools disclosed in WO 2009/090756 and Japanese Unexamined Patent Application Publication No. H10-197526, measurements are performed in accordance with a transmission method. In the analysis tools disclosed in WO 2009/090756 and Japanese Unexamined Patent Application Publication No. H10-197526, the amount of nicotinamide coenzymes are not directly measured at a wavelength in the ultraviolet region, as described above. In the analysis tool disclosed in WO 2009/090756, transport of the liquid sample is performed through application of pressure from a sample supply port. Analyzers for measuring such analysis tools are provided with a pump or the like for transporting a liquid sample, and hence tend to be large in size. In terms of achieving a smaller analyzer, it is preferable to use an analysis tool where a liquid sample is transported by capillarity, as in the analysis tool disclosed in Japanese Unexamined Patent Application Publication No. H10-197526.
In the analysis tool disclosed in Japanese Unexamined Patent Application Publication No. H10-197526, measurements relying on visible light are performed using a dry reagent that is disposed in a measurement chamber. In such an analysis tool, the dry reagent must be uniformly dissolved in the measurement chamber in order to perform measurements with good precision in accordance with the abovementioned recommended methods, in particular in accordance with a transmission method that utilizes ultraviolet light. Therefore, the dry reagent that is disposed in the measurement chamber must be smooth and free of cracks. However, it has been found that simple drying of reagent solutions in which nicotinamide coenzymes are dissolved result in impaired smoothness and formation of cracks. Unevenness arises in such dry reagents when dissolved without agitation, and hence achieving a target absorbance is difficult in cases where measurements are performed using light of the ultraviolet region. Moreover, cracks in the dry reagent result in bubbles that get caught in the cracks when a liquid sample flows into the measurement chamber. Bubbles present in such photometric sections are one cause of error in measured values.